Calcium‐Stimulated Adenosine Triphosphatases in Synaptic Membranes

Abstract

We have investigated the properties of several ATPases present in synaptic membrane preparations from the cerebral cortex of rat. In addition to the intrinsic (Na⁺+ K⁺)–ATPase and a low level of contaminating Mg²⁺–ATPase of mitochondrial origin, both of which could be controlled by the addition of ouabain and azide, respectively, four activities were studied: (1) a Mg²⁺–ATPase; (2) a Mg²⁺–independent activity requiring Ca²⁺ ions at high concentrations; (3) a (Ca²⁺+ Mg²⁺)–ATPase with a high affinity for Ca²⁺, which was enhanced further (4) by the inclusion of calmodulin (33 DM for half–maximal activity). In the presence of 0.5 mM–EGTA in the buffer used, half saturation for these respective metal ions was observed at 0.9 mM for (1), 1.0 DIM for (2), and –0.3 mM for (3) and (4); the latter values correspond to concentrations of free Ca²⁺ of 0.38 and 0.18 /XM for (3) and (4), respectively. The level of activities observed, all in nmol × min⁻¹× mg⁻′, under optimal conditions at 37°C, was in a number of preparations (n in parenthesis): for (1) 446 ± 19 (19); for (2) 362 ± 18 (3); for (3) 87 ± 13 (12); and for (4) 161 ± 29 (12). The (Ca²⁺+ Mg²⁺)-ATPase, both in the presence and absence of calmodulin, could be inhibited specifically by a number of agents (approximate I_0.5 in parentheses) which, at these concentrations, showed little or no potency against the other activities; among them were vanadate (≥10 μm), La³⁺ (75 μm), trifluoperazine, and other phenothiazines (50μm). These properties suggest that the (Ca²⁺+ Mg²⁺)-ATPase described may be responsible for calcium transport across one (or more) of the several membranes present in nerve endings and contained in the preparation used.